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Immobilized enzymes in bioprocess

S. F. D’Souza

Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay,

Mumbai 400 085, India

Immobilization of biocatalysts helps in their economic reuse and in the development of continuous bioprocesses. Biocatalysts can be immobilized either using the isolatedenzymes or the whole cells. Immobilization often stabilizes structure of the enzymes,thereby allowing their applications even under harsh environmental conditions of pH,temperature and organic solvents, and thus enable their uses at high temperatures innonaqueous enzymology, and in the fabrication of biosensor probes. In the future,development of techniques for the immobilization of multienzymes along with cofactor regeneration and retention system can be gainfully exploited in developing biochemicalprocesses involving complex chemical conversions. The present review outlines some of the above aspects, and delineates the present status and future potentials of immobilizedenzymes and nonviable cells in the emerging biotech industries.

Biotechnology is currently considered as a useful alternative to conventionalprocess technology in industrial and analytical fields. This is mainly because,unlike the chemical catalysts, the biological systems have the advantages of accomplishing complex chemical conversions under mild environmentalconditions with high specificity and efficiency. Biological systems help iningredient substitution, processing aid substitution, more efficient processing, lessundesirable products, increased plant capacity, increased product yields, andimproved or unique products. The variety of chemical transformations catalysedby enzymes has made these catalysts a prime target of exploitation by theemerging biotech industries.

Despite these advantages, the use of enzymes in industrial applications has beenlimited by several factors, mainly the high cost of the enzymes, their instability,and availability in small amounts. Also the enzymes are soluble in aqueous mediaand it is difficult and expensive to recover them from reactor effluents at the endof the catalytic process. This restricts the use of soluble enzymes to batchoperations, followed by disposal of the spent enzyme-containing solvent. Over thelast few decades, intense research in the area of enzyme technology hasprovided many approaches that facilitate their practical applications. Among them,the newer technological developments in the field of immobilized biocatalysts canoffer the possibility of a wider and more economical exploitation of biocatalysts in

industry, waste treatment, medicine, and in the development of bioprocessmonitoring devices like the biosensor.

Immobilization means associating the biocatalysts with an insoluble matrix, sothat it can be retained in a proper reactor geometry for its economic reuse under stabilized conditions. Immobilization thus allows, by essence, to decouple theenzyme location from the flow of the liquid carrying the reagents and products.Immobilization helps in the development of continuous processes allowing more



economic organization of the operations, automation, decrease of labour, andinvestment/capacity ratio. Immobilized biocatalysts offer several other advantages, notable among them is the availability of the product in greater purity. Purity of the product is very crucial in food processing and pharmaceutical

industry since contamination could cause serious toxicological, sensory, or immunological problems. The other major advantages include greater control over enzymatic reaction as well as high volumetric productivity with lower residencetime, which are of great significance in the food industry, specially in the treatmentof perishable commodities as well as in other applications involving labilesubstrates, intermediates or products1.

Immobilized nonviable cells as an economical source of enzymes

Biocatalysts can be immobilized using either the isolated enzymes or the wholecells or cellular organelles. Immobilization of whole cells has been shown to be a

better alternative to immobilization of isolated enzymes2–4

. Doing so avoids thelengthy and expensive operations of enzyme purification, preserves the enzymein its natural environment thus protecting it from inactivation either duringimmobilization or its subsequent use in continuous system. It may also provide amultipurpose catalyst, specially when the process requires the participation of number of enzymes in sequence. The major limitations which may need to beaddressed while using such cells are the diffusion of substrate and productsthrough the cell wall, and unwanted side reactions due to the presence of other enzymes. The cells can be immobilized either in a viable or a nonviable form.Immobilized nonviable cell preparations, which are normally obtained bypermeabilizing the intact cells, for the expression of intracellular activity are useful

for simple processes that require single-enzyme with no requirement for cofactor regeneration, like hydrolysis of sucrose or lactose2–4. On the other handimmobilized viable cells, which serve as ‘controlled catalytic biomass’, haveopened new avenues for continuous fermentation on heterogeneous catalysisbasis by serving as self-proliferating biocatalysts4,5. The present review will focusmainly on immobilized isolated enzyme systems, and the use of immobilizednonviable cells as a source of enzymes. For the aspects on the immobilizedviable cell systems, the readers can refer to another article in this issue. Some of the aspects on the applications of immobilized viable cells in biochemical processdevelopment and monitoring have also been reviewed recently6.

Most of the enzymes used at industrial scale are normally the extracellular enzymes produced by the microbes. This has been mainly due to their ease of isolation as crude enzymes from the fermentation broth. Moreover, theextracellular enzymes are more stable to external environmental perturbationscompared to the intracellular enzymes. However, over 90% of the enzymesproduced by a cell are intracellular. The economic exploitation of these, having avariety of biochemical potentials, has been limited in view of the high costinvolved in their isolation. Also, compared to extracellular enzymes, the



intracellular enzymes are more labile. Delicate and expensive separation methodsare required to release the enzymes undamaged from the cell, and to isolatethem. This increases the labour and the cost of the enzyme. These problemscould now be obviated by the use of permeabilized cells as a source of enzyme.

Permeabilization of the cells removes the barrier for the free diffusion of thesubstrate/product across the cell membrane, and also empties the cell of most of the small molecular weight cofactors, etc., thus minimizing the unwanted sidereactions. Side reactions, which can occur due to the presence of other enzymesin a cell, can also be minimized by inactivating such enzymes prior to or after immobilization7,8. Such permeabilized cells, which are often referred to asnonviable or nongrowing cells, can be exploited in an immobilized form as a veryeconomical source of intracellular enzyme for simple bioconversions likehydrolysis, isomerization and oxidation reactions that do not need a cofactor-regeneration system4. The decision to immobilize cells either in a viable or nonviable form is very important and depends on their ultimate application. Thus

the permeabilized K. fragilis cells, which are nonviable, convert lactose in milk toglucose and galactose9, whereas the viable (nonpermeabilized) cells convert thelactose to ethanol and are useful in the complete desugaration of milk10.

A variety of physical, chemical, and enzymatic techniques have been developedfor the permeabilization of cells9,11–16. Some techniques like entrapment inpolyacrylamide itself have been shown to result in permeabilization of the cells 11.A few typical examples of immobilized enzyme preparation obtained usingpermeabilized cells are listed in Table 1 (refs 17–30). It is evident that most of themajor industrially important intracellular enzymes can now be immobilized usingwhole cells.

Techniques and supports for immobilization

A large number of techniques and supports are now available for theimmobilization of enzymes or cells on a variety of natural and synthetic supports.The choice of the support as well as the technique depends on the nature of theenzyme, nature of the substrate and its ultimate application. Therefore, it will notbe possible to suggest any universal means of immobilization. It can only be saidthat the search must continue for matrices which provide facile, secureimmobilization with good interaction with substrates, and which conform in shape,size, density and so on to the use for which they are intended. Care has to be

taken to select the support materials as well as the reagents used for immobilization, which have GRAS status, particularly when their ultimateapplications are in the food processing and pharmaceutical industries.Macromolecular, colloidal, viscous, sticky, dense or particulate food constituentsor waste streams also limit the choice of reactor and support geometries.Commercial success has been achieved when support materials have beenchosen for their flow properties, low cost, nontoxicity, maximum biocatalystsloading while retaining desirable flow characteristics, operational durability, ease



of availability, and ease of immobilization31. The variety of techniques andsupports investigated for the immobilization have been reviewed in a number of articles and books1–4,32,33. The objective of the preceding part of the paper is toreview briefly, in the light of current developments, some approaches that have

been used for the immobilization of biocatalysts with reference to the studiescarried out in the author’s laboratory as well as few others.

Techniques for immobilization have been broadly classified into four categories,namely entrapment, covalent binding, cross-linking and adsorption. A combinationof one or more of these techniques has also been investigated. It must beemphasized that in terms of economy of a process, both the activity and theoperational stability of the biocatalysts are important. They determine itsproductivity, which is the activity integrated over the operational time.

Entrapment

Entrapment has been extensively used for the immobilization of cells, but not for enzymes. The major limitation of this technique for the immobilization of enzymesis the possible slow leakage during continuous use in view of the small molecular size compared tothe cells. Biocatalysts have been entrapped in natural polymers like agar, agaroseand gelatine through thermoreversal polymerization, but in alginate andcarrageenan by ionotropic gelation3,4,33. A number of synthetic polymers have alsobeen investigated. Notable among them are the photo-crosslinkable resins,

polyurethane prepolymers34

, and acrylic polymers like polyacrylamide17,24

. Amongthese, the most widespread matrix made from monomeric precursors is thepolyacrylamide gel. Polyacrylamide may not be a useful support for use in foodindustry in view of its toxicity, but can have potentials in the treatment of wasteand in the fabrication of analytical devices containing biocatalysts. One of themajor limitations of entrapment technique is the diffusional limitation as well asthe steric hindrance, especially when the macromolecular substrates like starchand proteins are used. Diffusional problems can be minimized by entrapment infine fibres of cellulose acetate or other synthetic materials32 or by using an openpore matrix35. Recently, the development of so-called hydrogels andthermoreactive water-soluble polymers, like the albumin-poly (ethylene glycol)

hydrogel, have attracted attention in the field of biotechnology36

. In the area of health care, they offer new avenues for enzyme immobilization. Such gels with awater content of about 96% provide a microenvironment for the immobilizedenzyme close to that of the soluble enzyme with minimal diffusional restrictions37.



Covalent binding

Covalent binding is an extensively used technique for the immobilization of enzymes, though it is not a good technique for the immobilization of cells. Thefunctional groups extensively investigated are the amino, carboxyl, and thephenolic group of tyrosine1,32. Enzymes are covalently linked to the supportthrough the functional groups in the enzymes, which are not essential for thecatalytic activity. It is often advisable to carry out the immobilization in thepresence of its substrate or a competitive inhibitor so as to protect the active site.The covalent binding should also be optimized so as not to alter itsconformational flexibility. Some of these problems however, can be obviated bycovalent bonding through the carbohydrate moiety when a glycoprotein is

concerned. A number of industrially useful enzymes are glycoproteins wherein thecarbohydrate moiety may not be essential for its activity. In general, functionalaldehyde group can be introduced in a glycoprotein by oxidizing the carbohydratemoiety by periodate oxidation without significantly affecting the enzyme activity.The enzyme could then be covalently linked to a support containing an alky aminegroup through Schiffs base reaction. Enzymes like glucose oxidase, peroxidase,invertase, etc. have been immobilized using this technique32,38,39. Covalent bindinghas been extensively investigated using inorganic supports. Enzymes covalently



bound to inorganic supports have been used in the industry 40. Enzymes have alsobeen bound to synthetic membranes, thus integrating biconversion anddownstream processing41,42. Large-scale processes using such an approach havebeen demonstrated for the preparation of invert sugar using invertase43.

Cross-linking

Biocatalysts can also be immobilized through chemical cross-linking using homo-as well as heterobifunctional cross-linking agents1. Among these, glutaraldehydewhich interacts with the amino groups through a base reaction has beenextensively used in view of its GRAS status, low cost, high efficiency, andstability44. The enzymes or the cells have been normally cross-linked in thepresence of an inert protein like gelatine, albumin, and collagen4,32. Studies fromour laboratory have shown the use of raw hen egg white as an economic, easilyavailable novel proteinic support rich in lysozyme for the immobilization of

enzyme or nonviable cells either in a powder 45–47

, bead48

or highly porous foam49,50

form, using glutaraldehyde as the cross-linker. The unique feature of this supportis the large concentration of lysozyme naturally present in hen egg white whichgets co-immobilized, thus imparting the bacteriolytic property to the support49,51.Adsorption followed by cross-linking has also been used for the immobilization of enzymes. The technique of cross-linking in the presence of an inert protein canbe applied to either enzymes or cells. The technique can also be used for theimmobilization of enzymes by cross-linking the cell homogenates52. Osmoticstabilization of cellular organelles53 or halophilic cells15 prior to immobilizationusing cross-linkers has also shown promise.

Adsorption

This is perhaps the simplest of all the techniques and one which does not grosslyalter the activity of the bound enzyme. In case of enzymes immobilized throughionic interactions, adsorption and desorption of the enzyme depends on thebasicity of the ion exchanger. Moreover, a dynamic equilibrium is normallyobserved between the adsorbed enzyme and the support which is often affectedby pH as well as the ionic strength of the surrounding medium. This property of reversibility of binding has often been used for the economic recovery of thesupport. This has been successfully adapted in industry for the resolution of racemic mixtures of amino acids, using amino acid acylase1,3. A variety of

commercially available ion exchangers have been investigated for this purpose32

.One of the techniques, which has gained importance more recently, is the use of polyethylenimine for imparting polycationic characteristics to many of the neutralsupports based on cellulose or inorganic materials54. Enzymes with low pI, likeinvertase55, urease56, glucose oxidase57, catalase57, and other enzymes54 havebeen bound through adsorption followed by cross-linking on polyethylenimine-coated supports.



Immobilization of enzymes through hydrophobic interaction has also shownpromise58,59. One of the important features of this technique, which is of greatsignificance, is that, unlike ionic binding, hydrophobic interactions are usuallystabilized by high ionic concentrations, thus enabling the use of high

concentrations of substrates as desired in an industrial process without the fear of desorption. Other types of strong interactive binding techniques have also beenreported for the reversible immobilization of enzymes. A typical example is theimmobilization of soybean b -amylase on phenyl-boronate agarose, which can bereversed for the recovery of the support using sorbitol60. Varieties of biospecificinteractions have also been investigated for the reversible immobilization of enzymes by adsorption. Enzymes like acetyl choline esterase, ascorbic acidoxidase, invertase, peroxidase, glucose oxidase, etc. have been immobilized bybiospecific-reversible immobilization on lectin-bound supports61,62 and invertase,using polyclonal antiinvertase antibodies63.

Traditional enzyme immobilization procedures involve isolation of the enzyme,followed by use of several steps for the immobilization. Costs of enzymepurification, the immobilization procedure, bioreactor operational stability, andbioreactor regeneration are the major factors that determine the cost of abioreactor process. Development of techniques for the simultaneous isolation andimmobilization of enzymes from crude extracts has obviated these problems.Some typical examples include the immobilization of a streptavidin-b-galactosidase fusion protein expressed in Escherichia coli and bioselectivelyadsorbed from a crude cell lysate to biotin which is covalently immobilized oncontrolled pore glass64, and the simultaneous purification and immobilization of D-amino acid oxidase from Trigonopsis variabilis cell lysate adsorbed on phenyl

sepharose

59

. The technique can have future potentials especially in thedownstream processing and immobilization of enzyme/proteins obtained byrecombinant DNA technology.

Techniques for the adhesion of whole cells on polymeric surfaces are alsocurrently gaining considerable importance. The major advantage for the cellsimmobilized through adhesion is reduction or elimination of the mass transfer problems associated with the commonly used gel entrapment method. Thetechnique of immobilization usually being followed is the microbial colonization byrecycling of the cell suspension along with nutrients, such that a biofilm isgradually formed. This often results in the immobilization of cells in a viable form

for use in heterogeneous fermentations

65

. Useful techniques have beendeveloped also for the immobilization of nonviable cells to be used as an enzymesource for simple chemical conversions. Notable among them include treating thecells or the support with trivalent metal ions like Al3+ or Fe3+ or charged colloidalparticles66, and use of polycationic polymers like chitosan67. Novel techniqueshave been developed to adhere cells strongly on a variety of polymeric surfacesincluding glass68, cotton cloth69, cotton threads19, and other synthetic andinorganic surfaces using polyethylenimine70. This technique has also been



recently used for the simultaneous filtration and immobilization of cells from aflowing suspension, thus integrating downstream processing with bioprocessing71.This technique may have future potentials for the immobilization of cells for foodapplications. Polyethylenimine is nontoxic and the United States of America Food

and Drug Administration has permitted its use as a direct food additive under theFood Drug and Cosmetic Act54.

Immobilized enzymes in organic solvents

In recent years, much research has centered on the conduct of enzyme reactionsin organic solvents. It is now well established that hydrolytic enzymes cancatalyse esterification and transesterification reactions in monophasic organicsolvents and in water-organic biphasic systems. Conventional immobilizedenzymes are principally used to facilitate catalysts recovery. Even thoughenzymes by themselves are insoluble in organic solvents, in addition to others,

the prime importance to the use of enzymes in organic media is the necessity toavoid enzyme deactivation or denaturation. A number of enzymes which are usedin organic solvents have been immobilized using a variety of techniques with aview to stabilizing them72. In systems containing enzymes immobilized on solidsupports and working in organic media, the support has a significant influence onthe total enzyme activity, and can also displace the reaction equilibrium(hydrolysis towards synthesis) due to the interaction of the support with the water molecules. Thus the choice of a suitable support material, proper water content,and the selection of the organic solvents are crucial for the use of immobilizedenzymes in organic media73,74. Another approach that has been explored is bychemical cross-linking of enzyme crystals, thereby stabilizing the crystalline lattice

and its constituent enzyme molecules, which result in forming highly concentratedimmobilized enzyme particles that can be lyophilized and stored indefinitely atroom temperature. Cross-linked enzyme crystals retain catalytic activity in harshconditions, including temperature and pH extremes, exogenous proteases, andexposure to organic or aqueous solvents75,76. Lyophilized cross-linked enzymecrystals can be reconstituted easily in these solvents as active monodispersesuspensions. Cross-linked enzyme crystals have shown promise in a variety of applications like the synthesis of aspartame, using thermolysin76, and for theresolution of chiral esters77. The techniques of immobilization can also beextended to obtain organic solvent-soluble enzymes by treating them withhydrophobic molecules like the lipids78.

Immobilization of enzymes for the fabrication of biosensors

Most of the techniques described above have been used for the immobilization of biocatalyst for biosensor applications79. The choice of the support and thetechnique for the preparation of membranes has been dictated by the lowdiffusional resistance of the membrane coupled with its ability to incorporateoptimal amount of enzyme per unit area. In this respect, stable membranes have



been prepared by binding glucose oxidase to cheese cloth in the fabrication of aglucose biosensor 80,81. Enzymes entrapped inside the reversed micelle have alsoshown promise in the fabrication of biosensors82. Cross-linked enzyme crystals(CLCs) described above provide their own supports and so achieve enzyme

concentration close to the theoretical packing limit in excess of even highlyconcentrated enzyme solutions. In view of this, CLCs are particularly attractive inbiosensor applications where the largest possible signal per unit volume is oftencritical75.

Sensors based on small transducer or thinner enzyme immobilized membranes(miniature biosensors) are also emerging. The development of molecular devicesincorporating a sophisticated and highly organized biological informationprocessing function, is a long-term goal of bioelectronics. For this purpose, it isnecessary in the future to develop suitable methods for microimmobilizing theproteins/enzymes into an organized array/pattern, as well as designing molecular

structures capable of performing the required function. A typical example is themicroimmobilization of proteins into organized patterns on a silicone wafer basedon a specific binding reaction between strepatavidin and biotin combined withphotolithography techniques83. Immobilized enzymes have also been used for various other analytical purposes. A recent development has been in obtaining astable dry immobilized enzyme, like acetylcholineesterase, on polystyrenemicrotitration plates for mass screening of its inhibitors in water and biologicalfluids84.

Immobilized multienzymes and enzyme-cellco-immobilizates

A common feature of metabolic pathways in intermediary metabolism is that theproduct of one enzyme in sequence is the substrate for the next and so forth. Oneof the advantages of such an arrangement is that a favourable high localconcentration of intermediates in the microenvironment of an enzyme system canbe created. Such an approach perhaps will be useful in the development of enzymatic processes requiring multiple enzymes. The enzymes in question canbe simultaneously bound to the same support or could be simultaneouslyimmobilized through entrapment1,34. Kinetic advantages of this system, such asreduction in the lag and significant enhancement in the final product, over mixedsoluble enzymes or immobilized and then mixed systems have been

demonstrated1,34,85

.

Often a cell may not contain all the necessary enzyme compliments for carryingout a required process. Thus Saccharomyces cerevisiae, a potential ethanolfermentor, cannot utilize lactose or starch since it lacks the enzyme b-galactosidase and amylase, respectively. There is considerable interest ingenetically modifying such cells for the expression of the deficient enzymes. Thesuccess will depend on the plasmid stability, and quite often on the ability of these



cells to secrete these enzymes for their action on the raw materials present in thegrowth medium. Recently, genetically engineered cells have been obtained whichcan not only produce the deficient enzyme but can also transport it to theperiplasmic place and naturally immobilize it86. Another more practical approach is

to immobilize the deficient enzyme directly on the cellwall. This approach which is termed as enzyme-cell co-immobilizate permits thetailoring of a whole cell for specific complex chemical conversion by combiningthe biochemical potential of both the cell and the additional enzyme from another source87. A few techniques developed in our laboratory include the binding of theglycoprotein glucose oxidase onto the microbial cell walls, using Con A85,88.Enzymes can also be immobilized on the microbial cell surfaces coated withpolyethylenimine through adsorption followed by cross-linking89. The differenttechniques for the preparation of enzyme–cell co-immobilizate, and their applications have been reviewed87.

Immobilized enzymes for the regeneration of cofactors

Enzyme technology has dealt mainly with simple reactions which do not requirecoenzymes. One of the major challenges for enzyme technologists is in theapplication of cofactor-dependent enzymes. Over 25% of the known enzymesrequire cofactors like NAD/NADH, ATP/ADP, etc. which, unlike the enzymes,undergo stoichiometric changes during a biochemical reaction. The living cellshave circumvented this problem either by introducing certain cofactor-recyclingenzyme systems in their metabolic pathways or through their recycling via theelectron transport system in the case of aerobic metabolism. In vitro applicationsof such enzymes will largely depend on developing efficient in vitro cofactor

recycling systems. These can be obtained by co-immobilizing another enzyme sothat it can recycle the cofactors, e.g. alcohol dehydrogenase in the presence of ethanol can be used for the recycling of NAD to NADH in a bioprocess catalysedby a NADH-dependent enzyme. Choice of the coupling enzyme for suchapplication has been made based on its ability to use an economical substratelike ethanol or formate (formate dehydrogenase) as well as one which resultspreferably in a volatile byproduct like CO2 or acetaldehyde so as to minimize thedownstream processing problems1,3,32,33. In addition to the recycling of thecofactor, other important criteria for development of cofactor-dependentbiochemical process is also the retention of cofactors. Currently methods areavailable for the covalent binding of the cofactors along with the enzymes on

polymeric supports or directly on enzymes or on soluble high molecular weightpolymers like dextran or polyethylene glycol32,33. Membrane reactors containingcontinuous cofactor regenerating systems have been used in the large-scaleproduction of sugar derivatives, and optically active a -hydroxy acids and a-amino acids90. Studies from our laboratory have shown that such cofactor regenerating system can be obtained by immobilization of permeabilized yeastcells as a source of alcohol dehydrogenase26,91. Multienzymes and cofactorsimmobilized within microcapsules have been used for the multi-step conversion of



liphophilic and lipophobic substrates92. A number of approaches have also beeninvestigated for the regeneration of ATP. One of these viable processes makesuse of acetate kinase which can phosphorylate ADP using acetyl phosphate asthe phosphate donor 32,33.

Applications of immobilized enzymes

The first industrial use of an immobilized enzyme is amino acid acylase byTanabe Seiyaku Company, Japan, for the resolution of recemic mixtures of chemically synthesized amino acids. Amino acid acylase catalyses thedeacetylation of the L form of the N -acetyl amino acids leaving unaltered the N -acetyl-d amino acid, that can be easily separated, racemized and recycled. Someof the immobilized preparations used for this purpose include enzymeimmobilized by ionic binding to DEAE-sephadex and the enzyme entrapped asmicrodroplets of its aqueous solution into fibres of cellulose triacetate by means of

fibre wet spinning developed by Snam Progetti. Rohm GmbH have immobilizedthis enzyme on macroporous beads made of flexiglass-like material93,94.

By far, the most important application of immobilized enzymes in industry is for the conversion of glucose syrups to high fructose syrups by the enzyme glucoseisomerase95. Some of the commercial preparations have been listed in Table 2. Itis evident that most of the commercial preparations use either the adsorption or the cross-linking technique. Application of glucose isomerase technology hasgained considerable importance, especially in nontropical countries that have



abundant starch raw material. Unlike these countries, in tropical countries likeIndia, where sugarcane cultivation is abundant, the high fructose syrups can beobtained by a simpler process of hydrolysis of sucrose using invertase. Comparedto sucrose, invert sugar has a higher humectancy, higher solubility and osmotic

pressure. Historically, invertase is perhaps the first reported enzyme in animmobilized form96. A large number of immobilized invertase systems have beenpatented97. The possible use of whole cells of yeast as a source of invertase wasdemonstrated by D’Souza and Nadkarni as early as 1978 (ref. 98). A systematicstudy has been carried out in our laboratory for the preparation of invert sugar using immobilized invertase or the whole cells of yeast17–19,38,45,55,68,69,71,98. Thesecomprehensive studies carried out on various aspects in our laboratory of utilizingimmobilized whole-yeast have resulted in an industrial process for the productionof invert sugar.

L-aspartic acid is widely used in medicines and as a food additive. The enzymeaspartase catalyses a one-step stereospecific addition of ammonia to the doublebond of fumaric acid. The enzymes have been immobilized using the whole cellsof Escherichia coli. This is considered as the first industrial application of animmobilized microbial cell. The initial process made use of polyacrylamideentrapment which was later substituted with the carragenan treated withglutaraldehyde and hexamethylenediamine. Kyowa Hakko Kogyo Co. uses

Duolite A7, a phenolformaldehyde resin, for adsorbing aspartase used in their continuous process99. Other firms include Mitsubishi Petrochemical Co.100 andPurification Engineering Inc101. Some of the firms, specially in Japan like TanabeSeiyaku and Kyowa Hakko, have used the immobilized fumarase for theproduction of malic acid (for pharmaceutical use)94. These processes make use of immobilized nonviable cells of Brevibacterium ammoniagenes or B. flavus as asource of fumarase. Malic acid is becoming of greater market interest as foodacidulant in competition with citric acid. Studies from our laboratory have shown



the possibility of using immobilized mitochondria as a source of fumarase6.

One of the major applications of immobilized biocatalysts in dairy industry is in the preparation of lactose-hydrolysed milk and whey, using b -galactosidase. A large population of lactoseintolerants can consume lactose-hydrolysed milk. This is of great significance in a country like

India where lactose intolerance is quite prevalent102. Lactose hydrolysis also enhances thesweetness and solubility of the sugars, and can find future potentials in preparation of a variety of dairy products. Lactose-hydrolysed whey may be used as a component of whey-basedbeverages, leavening agents, feed stuffs, or may be fermented to produce ethanol and yeast, thusconverting an inexpensive byproduct into a highly nutritious, good quality food ingredient99. Thefirst company to commercially hydrolyse lactose in milk by immobilized lactase was Centrale delLatte of Milan, Italy, utilizing the Snamprogetti technology. The process makes use of a neutrallactase from yeast entrapped in synthetic fibres103. Specialist Dairy Ingredients, a joint venturebetween the Milk Marketing Board of England and Wales and Corning, had set up an immobilizedb -galctosidase plant in North Wales for the production of lactose-hydrolysed whey. Unlike themilk, the acidic b -galactosidase of fungal origin has been used for this purpose31. Some of thecommercial b -galactosidase systems have been summarized in Table 3. An immobilizedpreparation obtained by cross-linking b -galactosidase in hen egg white (lyophilized dry powder)

has been used in our laboratory for the hydrolysis of lactose47

. A major problem in the large-scalecontinuous processing of milk using immobilized enzyme is the microbial contamination which hasnecessitated the introduction of intermittent sanitation steps. A co-immobilizate obtained bybinding of glucose oxidase on the microbial cell wall using Con A has been used to minimize thebacterial contamination during the continuous hydrolysis of lactose by the initiation of the naturallacto-peroxidase system in milk88. A novel technique for the removal of lactose by heterogeneousfermentation of the milk using immobilized viable cells of K. fragilis has also been developed10.

One of the major applications of immobilized enzymes in pharmaceutical industryis the production of 6-aminopenicillanic acid (6-APA) by the deacylation of theside chain in either penicillin G or V, using penicillin acylase (penicillinamidase)104. More than 50% of 6-APA produced today is enzymatically using the

immobilized route. One of the major reasons for its success is in obtaining a purer product, thereby minimizing the purification costs. The first setting up of industrialprocess for the production of 6-APA was in 1970s simultaneously by Squibb(USA), Astra (Sweden) and Riga Biochemical Plant (USSR). Currently, most of the pharmaceutical giants make use of this technology. A number of immobilizedsystems have been patented or commercially produced for penicillin acylasewhich make use of a variety of techniques either using the isolated enzyme or thewhole cells100,105,106

. This is also one of the major applications of the immobilizedenzyme technology in India. Similar approach has also been used for theproduction of 7-aminodeacetoxy-cephalosporanic acid, an intermediate in theproduction of semisynthetic cephalosporins.

Immobilized oxidoreductases are gaining considerable importance inbiotechnology to carry out synthetic transformations. Of particular significance inthis regard are oxidoreductase-mediated asymmetric synthesis of amino acids,steroids and other pharmaceuticals and a host of speciality chemicals. They playa major role in clinical diagnosis and other analytical applications like thebiosensors. Future applications for oxidoreductases can be in areas as diverse aspolymer synthesis, pollution control, and oxygenation of hydrocarbons107.



Immobilized glucose oxidase can find application in the production of gluconicacid, removal of oxygen from beverages, and in the removal of glucose from eggsprior to dehydration in order to prevent Maillard reaction. Studies carried out inthis direction in our laboratory have shown that glucose can be removed from

egg, using glucose oxidase and catalase which are co-immobilized either onpolycationic cotton cloth57 or in hen egg white foam matrix50. Alternatively, glucosecan also be removed by rapid heterogeneous fermentation of egg melange, usingimmobilized yeast108. Immobilized D-amino acid oxidase has been investigated for the production of keto acid analogues of the amino acids, which find application inthe management of chronic uremia. Keto acids can be obtained using either L- or D-amino acid oxidases. The use of D-amino acid oxidase has the advantage of simultaneous separation of natural L-isomer from DL-recemates along with theconversion of D-isomer to the corresponding keto acid which can then betransamina-ted in the body to give the L-amino acid. Of the severalmicroorganisms screened, the triangular yeast T . variabilis was found to be the

most potent source of D-amino acid oxidase with the ability to deaminate most of the D-amino acids109. The permeabilized cells entrapped either in radiationpolymerized acrylamide24 Ca-alginate23 or gelatin25 have shown promise in thepreparation of a -keto acids. Another interesting enzyme that can be usedprofitably in immobilized form is catalase for the destruction of hydrogen peroxideemployed in the cold sterilization of milk. A few reports are available on itsimmobilization using yeast cells11,22.

Lipase catalyses a series of different reactions. Although they were designed bynature to cleave the ester bonds of triacylglycerols (hydrolysis), lipase are alsoable to catalyse the reverse reaction under microaqueous conditions, viz.

formation of ester bonds between alcohol and carboxylic acid moieties. These twobasic processes can be combined in a sequential fashion to give rise to a set of reactions generally termed as interesterification. Immobilized lipases have beeninvestigated for both these processes. Lipases possess a variety of industrialpotentials starting from use in detergents; leather treatment controlled hydrolysisof milk fat for acceleration of cheese ripening; hydrolysis, glycerolysis andalcoholysis of bulk fats and oils; production of optically pure compounds, flavours,etc. Lipases are spontaneously soluble in aqueous phase but their naturalsubstrates (lipids) are not. Although use of proper organic solvents as anemulsifier helps in overcoming the problem of intimate contact between thesubstrate and enzyme, the practical use of lipases in such psuedohomogeneous

reactions poses technological difficulties. Varieties of approaches to solve these,using immobilized lipases, have recently been reviewed110.

Significant research has also been carried out on the immobilization and use of glucoamylase. This is an example of an immobilized enzyme that probably is notcompetitive with the free enzyme and hence has not found large-scale industrialapplication111. This is mainly because soluble enzyme is cheap and has beenused for over two decades in a very optimized process without technical



problems. Immobilization has also not found to significantly enhance thethermostability of amylase111. Immobilized renin or other proteases might allow for the continuous coagulation of milk for cheese manufacture112. One of the major limitations in the use of enzymes which act on macromolecular substrates or

particulate or colloidal substrates like starch or cellulose pectin or proteins hasbeen the low retention of their realistic activities with natural substrates due to thesteric hindrance. Efforts have been made to minimize these problems byattaching enzymes through spacer arms113. In this direction, application of tris(hydroxymethyl) phosphine as a coupling agent114 may have future potentials for the immobilization of enzymes which act on macromolecular substrates. Other problem, when particulate materials are used as the substrates for an enzyme, isdifficulty in the separation of the immobilized enzyme from the final mixture.Efforts have been made in this direction to magnetize the bicatalyst either bydirectly binding the enzyme on magnetic materials (magnetite or stainless steelpowder) or by co-entrapping magnetic material so that they can be recovered

using an external magnet98,115

. Magnetized biocatalysts also help in the fabricationof magnetofluidized bed reactor 116.

A variety of biologically active peptides are gaining importance in various fieldsincluding in pharmaceuti-cal industries and in food industries as sweeteners,flavourings, antioxidants and nutritional supplements. Proteases have emergedover the last two decades as powerful catalysts for the synthesis and modificationof peptides. The field of immobilized proteases may have a future role in thisarea117,118. One of the important large scale applications will be in the synthesis of peptide sweetener using immobilized enzymes like the thermolysin119. Proteolyticenzymes, such as subtilisin, a-chymotrypsin, papain, ficin or bromelain, which

have been immobilized by covalent binding, adsorption or cross-linking topolymeric supports are used (Bayer AG) to resolve A N -acyl-DL-phenylglycineester racemate, yielding N -acyl-D-esters or N -acyl-D-amides and N -acyl-L-acids100. Immobilized aminopeptidases have been used to separate DL-phenylgycinamide racemates100. SNAM-Progetti SpA-UK have used theimmobilized hydropyrimidine hydrolase to prepare D-carmamyl amino acids andthe corresponding D-amino acids from various substituted hydantoins100.

Conclusion

Achieving technical success is seldom sufficient, a lot of other factors have to befulfilled before industrial success is assured. The immobilized glucose isomerasewas adopted only when the market was ready and the technical problems weresolved. The same could be said about penicillin acylase. Immobilized enzymeshave to be very heat stable, work in a reliable optimized process system, and theproducts have to be cheap. If there is a cheap source of soluble enzyme in themarket or if other processes are well established, it is doubtful whether the newlydeveloped immobilized enzyme process can survive as in the case of glucoamylase. The cost considerations must take into account such factors as



cell production, matrix type and form, immobilization conditions, reactor design,and product purification. Economic and advantageous processes can be devisedbut their general industrial adoption will need time and encouragement. Pastexperience clearly suggests that we must learn to work with unpurified enzymes

like the whole cells or the cell homogenates. A significant future is expected for development of processes using multienzyme systems, specially those involvingcofactor regeneration for the production of high value compounds. The initialindustrial success in this direction is very encouraging90 and may hold a vastpotential for the future. The immobilized enzyme technology may also help in thefuture for integrating bioprocess with downstream processing with an effort toincrease the productivity while minimizing product recovery cost. Immobilizedenzyme technology may also be useful in nonaqueous enzymology, not only interms of stabilization of the biocatalysts but also in the development of continuousbioreactors. One of the rapidly emerging areas in the field of sensors120,121 is thedevelopment of biosensors for industrial process control wherein immobilized

enzyme technology will play a pivotal role. Stabilization of enzymes (heat) will becrucial for some of the applications. On the other hand, cold active enzymes alsomay gain potentials in certain biocoversions. Immobilized viable cells, which arediscussed elsewhere, will also have future potentials, specially in revolutionizingthe fermentation processes6. It is most likely that the future developments willcome from within the industry itself and the role of academic researchers willprobably remain to some extent as the laying of the theoretical foundations for this work and development of new approaches. Thus, there are interestingpossibilities within the field of immobilized enzymes and it is imminent that in thefuture many applications will be replaced by immobilized systems and many morenew systems will become technically as well as commercially feasible.

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1.

Membrane Technology

The University of Illinois at Urbana-Champaign has been conducting research in food

and biotechnology applications of membrane technology since 1975, in three major areas:

1. Process development2. Membrane characterization andevaluation

3. Engineering optimization.

Membrane technologies studied include reverse osmosis (RO), nanofiltration (NF),ultrafiltration (UF), microfiltration (MF), pervaporation (PV) and electrodialysis (ED).



In addition, a training program has been instituted, which includes a graduate course in

membrane technology, intensive industry-oriented short courses on campus and other

locations world wide and training of graduate students, post-doctoral fellows and visiting

scientists sponsored by international agencies and companies.

A modern 4000 square foot facility, which includes laboratories for chemical and

microbiological work, computing facilities, office space and a 2000 square foot pilot

plant, is devoted to this activity in the Agricultural Bioprocess Laboratory on the campus.

There are several bench-top test cells for membrane screening and characterization usingsamples as small as 25 mL,and several pilot scale units that are used for scale-up and

engineering studies that can handle several hundred liters per day. These include hollowfiber, spiral wound and tubular membrane designs in a variety of membrane materials(e.g., polymeric, ceramic, stainless steel). Pre- and post-treatment facilities are in adjacent

buildings with over 1150 square meters (12,500 square feet) of process equipment (spray

driers, drum driers, tray driers, freeze driers, evaporators, extruders, grinders,homogenizers, continuous centrifuges, milling machines, batch and continuous heat

exchangers for heating, cooling and freezing, etc.). This allows us to take an integrated process approach to projects if necessary.

http://www.fshn.uiuc.edu/courses/fshn460/index.html







The membrane technology and bioprocessing pilot plant at the University of Illinois. This

is one of three pilot plants in the department that total 1400 square meters (15,000



square feet).

1. PROCESS DEVELOPMENT

An example of this approach is our early work on the manufacture of purified functional

food ingredients and other value-added bioproducts from a variety of raw materials. For

example, the relatively high quantity and quality of protein and fat make soybeans a

valuable raw material for the manufacture of the protein isolates and other products thatcan be used in the formulation of a variety of nutritious foods. For optimum soybean

utilization, certain off-flavor factors and anti-nutritional compounds must be reduced or

eliminated, while maximizing yields and minimizing loss of functional properties. Wehave developed a process using ultrafiltration that meets these objectives. By careful

selection of membrane and operating conditions, the undesirable components can be

selectively removed. Since no heat is required and no phase change occurs, the process islow in energy and minimizes thermal damage to soybean constituents. Further, the whey

proteins, normally lost in conventional isolate manufacture, is retained in the final

product, which increases yields as well as improves functional properties. Full-fat soy protein concentrates are produced from whole soybeans after appropriate pre-treatment,

while soy isolates are produced from defatted soy meal.

Functional properties of these products have been studied and are superior to

conventional soy-products in some respects, especially solubility and dispersibility,emulsification and whipping. The emulsion stability at acidic pH makes it particularly

useful in mayonnaise-type products, and they can also be used to produce a smooth, tasty

and nutritious imitation milk. Publications on this topic

https://netfiles.uiuc.edu/mcheryan/www/soy.htm

https://netfiles.uiuc.edu/mcheryan/www/publicat.htm#pubsoy

https://netfiles.uiuc.edu/mcheryan/www/soy.htm

https://netfiles.uiuc.edu/mcheryan/www/publicat.htm#pubsoy



Corn (maize) protein isolates and concentrates can also be produced by membrane

technology. A combination of solvents such as ethanol to extract the zein and alkali to

extract the glutelin is used with the appropriate membranes. Corn protein hydrolyzateswith improved functional properties have been produced using enzymes and membrane

separations. Publications on thistopic

The binding of sunflower protein to polyphenols was studied using ultrafiltration. This

provided an understanding of the nature and extent of binding of undesirable

polyphenols, which was useful in developing a process to manufacture sunflower proteinisolates and concentrates.

Publications on this topic

Milk and cheese whey protein concentrates and isolates have been a focus for more than

two decades. Membrane technology allows the separation and isolation of dairy proteinsin a manner that frequently results in superior and unique functional properties. One of

the earliest successful applications of ultrafiltration, it is a good example of converting an

otherwise tremendous source of pollution into useful products. Cheese whey is notoriousfor severely fouling membranes. A vast amount of research over a period of 10 years hasresulted in a better understanding of the problem and methods to minimize these effects.

Today, membrane processing of cheese whey is a well-accepted standard unit operation

in the dairy industry around the world. Publications on this topic

Bioprocessing

Membranes can be valuable in biotechnology applications, especially downstream

processing. We have focused on four aspects:

• Harvesting of microbial cells: studies have been done on the influence of module

design (hollow fibers vs. tubular vs. pleated sheets vs. ceramics), type of microorganism (yeast, bacteria, fungi) and operating parameters on performance,

recovery of cells and fouling,

• Fractionation of fermentation broths and recovery of products,

• Cold sterilization of fermentation media,

• Design of high-performance bioreactors.

"Bioreactors" refer to equipment wherein biological substances undergo a chemical

change in the presence of a biological catalyst such as an enzyme or microorganism.

Traditionally, this has meant using "batch" reactors or fermenters, which hasdisadvantages such as high capital and labor costs, inconsistent products (batch to batch

variation), the expense of separating and inactivating the biocatalyst at the end of the process and low productivity (defined as amount of product per unit reactor volume per

unit time).

An alternate approach has been used in our laboratory at the University of Illinois for

designing high-performance bioreactors, specifically for enzymatic and microbial

https://netfiles.uiuc.edu/mcheryan/www/avcpro.htm#cope

https://netfiles.uiuc.edu/mcheryan/www/avcpro.htm

https://netfiles.uiuc.edu/mcheryan/www/publicat.htm#pubcorn

https://netfiles.uiuc.edu/mcheryan/www/publicat.htm#pubsun

https://netfiles.uiuc.edu/mcheryan/www/dairy.htm

https://netfiles.uiuc.edu/mcheryan/www/publicat.htm#pubdairy

https://netfiles.uiuc.edu/mcheryan/www/publicat.htm#pubbior

https://netfiles.uiuc.edu/mcheryan/www/avcpro.htm#cope

https://netfiles.uiuc.edu/mcheryan/www/avcpro.htm

https://netfiles.uiuc.edu/mcheryan/www/publicat.htm#pubcorn

https://netfiles.uiuc.edu/mcheryan/www/publicat.htm#pubsun

https://netfiles.uiuc.edu/mcheryan/www/dairy.htm

https://netfiles.uiuc.edu/mcheryan/www/publicat.htm#pubdairy

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performance of membrane systems (flux and rejection). Mathematical models, based on

semi-empirical and classic momentum and mass transfer correlations, are developed for

specific MF, UF, NF, RO and PV applications. Of special interest is the behavior of colloidal feed streams, which are typical of many food and biological systems, which

tend to give experimental fluxes much higher than predicted by classic mass transfer

models.

Two areas of special concern are membrane fouling (and cleaning) and energyconsumption characteristics of various membranes and modules. The fouling

phenomenon has been studied using model and real systems. X-ray analysis and electron

microscopy of the fouled membrane elucidated the nature and extent of the interactions between feed components and the membrane. This was valuable in developing feed pre-

treatments and recommendations to minimize membrane-solute interactions. Studies are

also being done on the effect of membrane module design and operating conditions onthe fouling phenomenon.

The ultimate goal is to design the optimum membrane process. We have a closerelationship with several membrane manufacturers and engineering companies. This

enables us to provide good estimates of capital and operating costs to project sponsors,with data obtained in our pilot plant using commercial scale modules in most cases. This

can substantially reduce the time and risk for potential users of the technology. Publications on membrane technology

Membrane processes in biotechnology and biochemical industry

The biotechnology industry, which originated in the late 1970s, has become one of the

emerging industry that draws the attention of the world, especially with the emergence of the genetic engineering as a means of producing medically important proteins, during the

1980s. Two of the major interest applications of membrane technology in the

biotechnology industry will be the separation & purification of the biochemical product,

as often known as Downstream Processing ; and the membrane bioreactor, whichdeveloped for the transformation of certain substrates by enzymes (i.e. biological

catalysts). Lots of literature has been published since the last ten years for these topic

which serve as a good reference is sited in the Reference.

• Downstream processing

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"Downstream Processing", a new key term given a decades ago, devotes towards the

science and engineering principles in separation and purification in this emerging

industry, has become a key issue to enhance the quality of the biochemical product. It is particularly important because it typically accounts for nearly three-fourths of the

manufacturing costs in this new industry and because reliable and effective purification

can be of the utmost important to the user. Membrane separation, together with the bioaffinity chromatography, liquid extraction and selective precipitation are the few

techniques in the bio-separations, which gain attention from both the industries and

researcher in order to upgrade the product quality of the biochemical industry.

Lots of study has been put in this area involving the most of the recovery of the biofuelsand the biochemicals. Throughout the available literature, the most useful review is

presented by Stephen A Leeper (1992), which compiles a large number of the previous

and current studies' data on the different types of biofuels and the biochemicals productrecovery, consisting of the usage of different types of membrane materials, membrane

processes, together with the operating parameters of the studies being carried out.

• Membrane bioreactor

Since its introduction in the 1970s, membrane bioreactor has granted a lot of attention

over the other conventional production processes is the possibility of a high enzymedensity and hence high space-time yields. Whereas downstream processing is usually

based on discontinuously operated microfiltration, membrane bioreactor are operated

continuously and are equipped with UF membranes. Two type of bioreactor designs are possible: dissolved enzymes, (as in used with the production of L-alanine from pyrurate)

or immobilized enzymes membrane.

Future Prospects

Membrane science began emerging as an independent technology only in the mid-1070s,and its engineering concepts still are being defined. Many developments that initially

evolved from government-sponsored fundamental studies are now successfully gaining

the interest of the industries as membrane separation has emerged as a feasible

technology.

As were noted by the US National Research Council, the technological frontiers of the

membrane technology should be concerned more in the developing of new membrane

materials and the identification of new ways of using permselective membranes.

New membrane materials to be used is still a big option in the research of this brand newtechnology, as most of the researchers are always intend to get a better improvement for

this separation process. Journal of Membrane Science serve as a good reference, where

lots of the new membrane materials research may be found.

For the latter, membrane-based hybrid system serve as a good example, as it is acombination of conventional unit operations and membrane separation processes, which



often results in separation processes that offer significant advantages over the exclusive

use of either component process. Such advantages may include more complete

separation, reduced energy requirement, lower capital cost, and lower production cost.Two good example of this hybrid system are the RO / evaporator hybrid system to

concentrate corn steep water, and a membrane / vapor-recompression hybrid process to

recover energy in hot, moist dryer exhaust. Studies has also been carried out and proventhat these hybrid system did perform a better of the washwater purification and reuse

pilot plant (HUMEF) which has been successful installed in Eindhoven pumping station,

the Netherlands.

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